The Study Of Glycosphingolipid GM1 Mediating The Proliferation And Migration In Mammary Epithelial Cells

Glycosphingolipids(GSLs),which consist of a hydrophobic ceramide backbone and a hydrophilic carbohydrate residue,are an important type of glycolipid expressed in surface membranes of all animal cells.GM1,a sialic acid-containing subtype of GSLs,are play essential roles in maintenance of plasma membrane stability,in regulation of numerous cellular processes(including proliferation,adhesion,and recognition),and in modulation of signal transduction pathways.GM1 is also involved in the development and progression of cancer.However,the regulatory role of GM1 on mammary epithelial cell proliferation and migration and the associated molecular mechanisms still need clarified,which may provide insight into the role of GM1 in the development of breast cancer and provide an experimental basis for the treatment of breast cancer.In this study,we analyzed the effects of exogenous addition and endogenous alteration of GM1 on proliferation and migration in mammary epithelial cells,and proposed the possible mechanism of GM1 regulating the proliferation and migration of mammary epithelial cells.The main results are as follows:(1)The effect of exogenous addition of GM1 on the proliferation of mammary epithelial cells at different cell densities.Using EdU incorporation assays,Western blot,and flow cytometry,we confirmed the contact inhibition of growth in human mammary epithelial cells MCF-10 A,MCF-7,MDA-MB-231,BT-549,and SK-BR-3 when growing at high density.The results of flow cytometry and TLC assays revealed that GM1 expression was increased significantly in high-density cells.Exogenous addition of GM1 could inhibit cell proliferation and inactivate the EGFR signaling pathway in high-density cells,but not in normal-density cells.(2)The effects of knockdown and overexpression of GM1 on the proliferation of mammary epithelial cells.GM1 knockdown and GM1 overexpression stable transfected cell lines were constructed by lentivirus vectors.Overexpression of GM1 could inhibit cell proliferation and inactivate the EGFR signaling pathway in high-density cells,while knockdown of GM1 showed the opposite results.However,overexpression or knockdown of GM1 had no significant effect on cell proliferation and activation of EGFR signaling pathway in normal-density cells.(3)The molecular mechanism of GM1 inhibiting EGFR activation.Coimmunoprecipitation,OptiPrep density gradient and immunofluorescence staining revealed that the distribution of EGFR translocated from GEM to caveolae in high-density cells compared to the cells grown at normal density.Based on our observation,we propose that GM1 suppressed EGFR signaling and promoted contact inhibition of growth by translacoting the EGFR from GEM domain to caveolae domain.(4)The effect of GM1 on mammary epithelial cell migration.Overexpression of GM1 was found to induce EMT-like changes in MCF-10 A cells.Hanging drop aggregation assay and attachment assay confirmed that cell-cell adhesion was weakened and the adhesion between cells and extracellular matrix was enhanced in GM1 overexpression MCF-10 A cells.Transwell migration assays showed that overexpression of GM1 promoted cell migration.The overexpression of GM1 could further promote the TGF-β-induced EMT process.In summary,overexpression of GM1 increased migration and induced EMT-like changes in mammary epithelial cells.(5)The role of GM1 in exosome-mediated promotion of r cell migration.Western blot and transwell migration assay confirmed that the conditioned medium from MDA-MB-231 cells could increase the cell migration and induce EMT-related changes of MCF-10 A and MCF-7 cells,while overexpression of GM1 in MDA-MB-231 cells could enhance this effect of conditioned medium.Exosomes were extracted from conditioned medium by differential centrifugation.The purification of exosomes was verified by Western blot,nanoparticle tracking analysis,and transmission electron microscopy.The expression of GM1 in exosomes was confirmed by immunoelectron microscopy,density gradient centrifugation and flow cytometry,and overexpression of GM1 in donor cells could increase the content of GM1 in exosomes.Exosomes from MDA-MB-231 cells could increase the cell migration and induce EMT-related changes of MCF-10 A and MCF-7 cells,while overexpression of GM1 in MDAMB-231 cells could enhance this effect of exosmes.